ORIGINAL ARTICLE
The Clinical Respiratory Journal Journal
Imbalance of peripheral blood Th17 and Treg responses in patients with chronic obstructive pulmonary disease Huaying Wang1, Huajuan Ying1, Shi Wang1, Xiao Gu1, Yuesong Weng2, Weidong Peng1, Dajing Xia3 and Wanjun Yu1 1 Department of Respiratory and Critical Care Medicine, The Affiliated Yinzhou Hospital, College of Medicine, Ningbo University, Ningbo, Zhejiang, China 2 Clinical Laboratory, The First Hospital, Ningbo, Zhejiang, China 3 Institute of Immunology, College of Medicine, Zhejiang University, Hangzhou, China
Abstract Background: Autoimmune responses mediated by cluster of differentiation 4+ T cells may contribute to the development of chronic obstructive pulmonary disease (COPD). However, little is known about the frequency of peripheral blood Th17 cells and of regulatory T cells (Tregs) in Chinese patients with COPD. This study is aimed at determining the frequency of circulating Th17 and Tregs in patients with moderate and severe COPD, heavy smokers and healthy controls (HC). Method: The percentages of circulating T-helper type (Th)17 cells and Tregs were determined by flow cytometry in 32 patients with moderate COPD, 33 patients with severe COPD, 35 smokers, and 31 HC. The concentrations of serum Th17and Treg-related cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The levels of retinoic acid orphan receptor (ROR)-γt and Forkhead box p3 (Foxp3) mRNA transcripts in peripheral blood mononuclear cells were determined by real-time polymerase chain reaction. The potential correlation between the percentages of Th17 Tregs, serum cytokines and lung function was evaluated. Results: In comparison with that in the smokers and HC, significantly higher frequencies of Th17 cells and higher levels of ROR-γt mRNA transcripts and serum interleukin (IL)-17A, IL-6, IL-21, IL-22 and IL-23, but lower frequency of Tregs and lower levels of Foxp3 and serum IL-10 were detected in patients with moderate and severe COPD. The increased ratios of Th17 to Tregs were negatively correlated with the values of forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1) and FEV1/FVC. Conclusions: An imbalance of circulating Th17 cells and Tregs is associated with the deterioration of pulmonary function in patients with moderate and severe COPD.
Please cite this paper as: Wang H, Ying H, Wang S, S, Gu Gu X, X, Weng Weng Y, Y, Peng Peng W, W, Xia Xia D D and Yu W. Imbalance of peripheral blood Th17 and Treg Treg responses responses in in patients patients with with chronic obstructive 9: ••: 330–341. obstructivepulmonary pulmonarydisease. disease.Clin ClinRespir RespirJ 2015; J 2014; ••–••. DOI:10.1111/ crj.12147.
Conflict of interest The authors have stated explicitly that there are no conflicts of interest in connection with this article.
330 The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
Key words COPD – immune imbalance – pulmonary function – regulatory T cells – Th17 cells Correspondence Wanjun Yu, MD, Department of Respiratory and Critical Care Medicine, The Affiliated Yinzhou Hospital, College of Medicine, Ningbo University, No. 251, Baizhang Road, 315040 Ningbo, Zhejiang, China. Tel: 86-574-87016981 Fax: 86-574-87017272 email: [email protected] Received: 14 July 2013 Revision requested: 03 March 2014 Accepted: 04 April 2014 DOI:10.1111/crj.12147 Authorship and contributorship HW carried out the clinical study, performed flow cytometry analysis, enzyme-linked immunosorbent assay measurement and drafted the manuscript. HY, SW and YW collected clinical samples and carried out reserve transcription-polymerase chain reaction. XG and WP collected clinical samples and performed flow cytometry analysis. DX participated in the design of the study and performed statistical analysis. WY conceived of the study, interpreted the data and wrote the manuscript. All authors read and approved the final manuscript. Ethics Written informed consent was obtained from individual subjects, and the experimental protocol was approved by the Ethics and Research Committees of the Affiliated Yinzhou Hospital, College of Medicine, Ningbo University.
The Clinical Respiratory Journal (2015) • ISSN 1752-6981 1 C 2014 John Wiley & Sons Ltd V
Wang and et al.Tregs in COPD Th17
Abbreviations: COPD chronic obstructive pulmonary disease ELISA enzyme-linked immunosorbent assay FSC-A forward scatter FEV1 forced expiratory volume in 1 s Foxp3 Forkhead box P3 (the Treg-defining transcription factor) FVC forced vital capacity HC healthy controls ICS inhaled corticosteroid
Th17 and Tregs in COPD Wang et al.
IL interleukin PBMC peripheral blood mononuclear cell PMA phorbol 12-myristate 13-acetate PCR polymerase chain reaction ROR-γt retinoic acid orphan receptor (the Th17-defining transcription factor) RT-PCR reserve transcription-polymerase chain reaction TGF-β transforming growth factor β Th T-helper type Tregs regulatory T cells
Background Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation-mediated progressive and irreversible airflow obstruction (1). COPD is a dangerous disease with high morbidity and mortality worldwide (2). Currently, the pathogenic process of COPD is still not fully understood. Previous studies have shown that many factors contribute to the development and progression of COPD (3, 4). Systemic chronic inflammation, alveolar macrophages, cluster of differentiation (CD)8+ T lymphocytes, elevated levels of inflammatory cytokines and an impaired regulatory system are crucial for the development and progression of COPD (5–12). However, the precise role of different subsets of CD4+ T cells in the pathogenesis of COPD remains unclear (13). Therefore, further illustration of the pathogenic process of COPD will be of great significance in the management of patients with COPD. Activated CD4+ T cells can differentiate into different subsets of functional T cells, such as T-helper type (Th)1, Th2, Th17, Th22 and regulatory T cells (Tregs). Autoreactive CD4+ T cells recognizing self and smoking-modified lung antigens have implicated the pathogenic process of COPD (14–16). Th17 cells secrete pro-inflammatory cytokines, such as interleukin (IL)-17A, IL-17F, IL-21, IL-22 and IL-23. The development of Th17 cells is dependent on retinoic acid orphan receptor (ROR)-γt expression and positively regulated by IL-6, IL-1β and IL-23 as well as transforming growth factor β (TGF-β), but negatively regulated by IL-10. Th17 cells are crucial for the pathogenesis of several inflammatory and autoimmune diseases (17, 18). Previous studies have suggested that Th17 cells may participate in the inflammatory process of COPD in animal models and humans (19–21). However, whether Th17 cells also participate in the pathogenic process of COPD in Chinese patients, who have a history of heavy smoking and exposure to dangerous air pollution, has not been clarified. CD4+CD25+Foxp3+ Tregs can inhibit inflammation (22). Treg development is dependent on the Forkhead box p3 (Foxp3) expression and is positively regulated by The Clinical Respiratory Journal (2015) • ISSN 1752-6981 2
C 2014 John Wiley & Sons Ltd V
TGF-β. Functionally, Tregs secrete inhibitory IL-10 and TGF-β. The deficiency in the number and function of Tregs is associated with the development of autoimmune diseases. However, how Tregs regulate the development of COPD remains controversial. While a higher frequency of circulating Tregs was detected in patients with COPD, along with higher percentages of Th17 cells (19), lower percentages of CD4+CD25+ T cells were detected in another population of COPD patients (23). Currently, there is no information about the frequency of circulating Tregs in Chinese patients with COPD. Notably, a previous study indicated that the imbalance of Th17 and Tregs was associated with the smokingrelated COPD in an animal model (24). Given that chronic heavy smoking causes inflammation and inhibits Treg responses, it is unclear whether the imbalance of Th17 and Tregs can exist in Chinese COPD patients, who have a history of heavy smoking and commonly expose to air pollution. Furthermore, how the degrees of Th17 and Treg imbalance are associated with the pulmonary function in those patients has not been clarified. In this study, we characterized the frequency of peripheral blood Th17 and Tregs, the relative levels of ROR-γt and Foxp3 mRNA transcripts in peripheral blood samples, and measured the levels of serum IL-17A, IL-1β, IL-6, IL-10, IL-21, IL-22, IL-23 and TGF-β in 32 Chinese patients with moderate COPD, 33 with severe COPD, 31 heavy smokers and 35 healthy controls (HC). Furthermore, we analyzed the potential association of the values of Th17, Tregs, cytokines and pulmonary function in those patients.
Materials and methods Subjects A total of 65 patients with COPD diagnosed more than 1 year, 35 smokers and 31 HC with normal pulmonary function were recruited sequentially at the inpatient and outpatient services of the Affiliated Yinzhou Hospital, College of Medicine, Ningbo University from 2010 to 2012. Individual patients with COPD were diagnosed, according to the criteria, including the 331 The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
Th17 Wangand et al.Tregs in COPD
Wang et al. Th17 and Tregs in COPD
Table 1. The demographic and clinical characteristics of participants Measure
HC (n = 31)
Smokers (n = 35)
Moderate COPD (n = 32)
Severe COPD (n = 33)
Age (year) Gender (M/F) Body mass index (kg/m2) Tobacco (pack-year) Number of admissions (time/year) LOHS (day) Use of LAMA Use of LABA Inhaled corticosteroids FEV1 (L) FEV1 % predicted FVC (L) FVC % predicted FEV1/FVC (%) WBC (×109/L) Lymphocytes (%) Neutrophils (%) CD4+ T cells (%) CD8+ T cells (%)
64 ± 8 18/15 23 ± 5.1 – – – – – – 3.3 ± 0.3 103.6 ± 7.3 4.0 ± 0.2 102.7 ± 6.7 82.3 ± 6.1 6.6 ± 1.2 30.3 ± 4.5 64.5 ± 7.2 37.0 ± 5.1 28.8 ± 4.8
67 ± 10 32/3 24.3 ± 4.9 37 (10,70) – – – – – 2.7 ± 0.2* 98.3 ± 7.7* 3.4 ± 0.15* 96.7 ± 7.8* 81.6 ± 5.8 5.9 ± 1.7 33.2 ± 3.8 63.4 ± 6.9 40.2 ± 6.8 26.4 ± 4.1
66 ± 7 26/6 24.0 ± 5.5 44 (10,82) 1.7 ± 0.7 10.4 ± 3.5 3 29 29 1.3 ± 0.2**## 53.4 ± 8.8**## 2.9 ± 0.2**## 81.4 ± 9.3**## 43.8 ± 4.4**## 6.1 ± 0.9 29.8 ± 5.1 67.8 ± 7.3 46.1 ± 6.2**## 20.6 ± 4.7**##
68 ± 7 28/5 23.6 ± 3.8 41 (18,90) 1.8 ± 0.8 13.6 ± 4.8▲▲ 2 26 26 0.7 ± 0.1**##▲▲ 36.4 ± 7.2**##▲▲ 2.7 ± 0.2**## 79.9 ± 9.6**## 26.7 ± 3.2**##▲▲ 6.5 ± 1.3 34.1 ± 3.8 60.4 ± 6.8 50.3 ± 7.5**##▲▲ 16.4 ± 3.9**##▲▲
The therapy of ICS + LABA used by COPD patients in this study is salmeterol xinafoate and fluticasone propionetae powder for inhalation or budesonide and formoterol fumarate powder for inhalation. The LAMA used by COPD patients in this study is tiotropium bromide powder for inhalation. All data expressed as mean ± standard error of the mean or median and range. *P < 0.05 and **P < 0.01 vs the HC group. # P < 0.05 and ##P < 0.01 vs the smokers. ▲ P < 0.05 and ▲▲P < 0.01 vs the moderate COPD group. CD, cluster of differentiation; COPD, chronic obstructive pulmonary disease; F, female; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; HC, healthy controls; ICS, inhaled corticosteroid; LABA, long-acting beta agonist; LAMA, long-acting muscarinic antagonist; LOHS, length of hospital stay; M, male; WBC, white blood cell.
history of tobacco smoking, clinical symptoms and impaired pulmonary function of forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) postbronchodilator
The Clinical Respiratory Journal Journal
Imbalance of peripheral blood Th17 and Treg responses in patients with chronic obstructive pulmonary disease Huaying Wang1, Huajuan Ying1, Shi Wang1, Xiao Gu1, Yuesong Weng2, Weidong Peng1, Dajing Xia3 and Wanjun Yu1 1 Department of Respiratory and Critical Care Medicine, The Affiliated Yinzhou Hospital, College of Medicine, Ningbo University, Ningbo, Zhejiang, China 2 Clinical Laboratory, The First Hospital, Ningbo, Zhejiang, China 3 Institute of Immunology, College of Medicine, Zhejiang University, Hangzhou, China
Abstract Background: Autoimmune responses mediated by cluster of differentiation 4+ T cells may contribute to the development of chronic obstructive pulmonary disease (COPD). However, little is known about the frequency of peripheral blood Th17 cells and of regulatory T cells (Tregs) in Chinese patients with COPD. This study is aimed at determining the frequency of circulating Th17 and Tregs in patients with moderate and severe COPD, heavy smokers and healthy controls (HC). Method: The percentages of circulating T-helper type (Th)17 cells and Tregs were determined by flow cytometry in 32 patients with moderate COPD, 33 patients with severe COPD, 35 smokers, and 31 HC. The concentrations of serum Th17and Treg-related cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The levels of retinoic acid orphan receptor (ROR)-γt and Forkhead box p3 (Foxp3) mRNA transcripts in peripheral blood mononuclear cells were determined by real-time polymerase chain reaction. The potential correlation between the percentages of Th17 Tregs, serum cytokines and lung function was evaluated. Results: In comparison with that in the smokers and HC, significantly higher frequencies of Th17 cells and higher levels of ROR-γt mRNA transcripts and serum interleukin (IL)-17A, IL-6, IL-21, IL-22 and IL-23, but lower frequency of Tregs and lower levels of Foxp3 and serum IL-10 were detected in patients with moderate and severe COPD. The increased ratios of Th17 to Tregs were negatively correlated with the values of forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1) and FEV1/FVC. Conclusions: An imbalance of circulating Th17 cells and Tregs is associated with the deterioration of pulmonary function in patients with moderate and severe COPD.
Please cite this paper as: Wang H, Ying H, Wang S, S, Gu Gu X, X, Weng Weng Y, Y, Peng Peng W, W, Xia Xia D D and Yu W. Imbalance of peripheral blood Th17 and Treg Treg responses responses in in patients patients with with chronic obstructive 9: ••: 330–341. obstructivepulmonary pulmonarydisease. disease.Clin ClinRespir RespirJ 2015; J 2014; ••–••. DOI:10.1111/ crj.12147.
Conflict of interest The authors have stated explicitly that there are no conflicts of interest in connection with this article.
330 The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
Key words COPD – immune imbalance – pulmonary function – regulatory T cells – Th17 cells Correspondence Wanjun Yu, MD, Department of Respiratory and Critical Care Medicine, The Affiliated Yinzhou Hospital, College of Medicine, Ningbo University, No. 251, Baizhang Road, 315040 Ningbo, Zhejiang, China. Tel: 86-574-87016981 Fax: 86-574-87017272 email: [email protected] Received: 14 July 2013 Revision requested: 03 March 2014 Accepted: 04 April 2014 DOI:10.1111/crj.12147 Authorship and contributorship HW carried out the clinical study, performed flow cytometry analysis, enzyme-linked immunosorbent assay measurement and drafted the manuscript. HY, SW and YW collected clinical samples and carried out reserve transcription-polymerase chain reaction. XG and WP collected clinical samples and performed flow cytometry analysis. DX participated in the design of the study and performed statistical analysis. WY conceived of the study, interpreted the data and wrote the manuscript. All authors read and approved the final manuscript. Ethics Written informed consent was obtained from individual subjects, and the experimental protocol was approved by the Ethics and Research Committees of the Affiliated Yinzhou Hospital, College of Medicine, Ningbo University.
The Clinical Respiratory Journal (2015) • ISSN 1752-6981 1 C 2014 John Wiley & Sons Ltd V
Wang and et al.Tregs in COPD Th17
Abbreviations: COPD chronic obstructive pulmonary disease ELISA enzyme-linked immunosorbent assay FSC-A forward scatter FEV1 forced expiratory volume in 1 s Foxp3 Forkhead box P3 (the Treg-defining transcription factor) FVC forced vital capacity HC healthy controls ICS inhaled corticosteroid
Th17 and Tregs in COPD Wang et al.
IL interleukin PBMC peripheral blood mononuclear cell PMA phorbol 12-myristate 13-acetate PCR polymerase chain reaction ROR-γt retinoic acid orphan receptor (the Th17-defining transcription factor) RT-PCR reserve transcription-polymerase chain reaction TGF-β transforming growth factor β Th T-helper type Tregs regulatory T cells
Background Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation-mediated progressive and irreversible airflow obstruction (1). COPD is a dangerous disease with high morbidity and mortality worldwide (2). Currently, the pathogenic process of COPD is still not fully understood. Previous studies have shown that many factors contribute to the development and progression of COPD (3, 4). Systemic chronic inflammation, alveolar macrophages, cluster of differentiation (CD)8+ T lymphocytes, elevated levels of inflammatory cytokines and an impaired regulatory system are crucial for the development and progression of COPD (5–12). However, the precise role of different subsets of CD4+ T cells in the pathogenesis of COPD remains unclear (13). Therefore, further illustration of the pathogenic process of COPD will be of great significance in the management of patients with COPD. Activated CD4+ T cells can differentiate into different subsets of functional T cells, such as T-helper type (Th)1, Th2, Th17, Th22 and regulatory T cells (Tregs). Autoreactive CD4+ T cells recognizing self and smoking-modified lung antigens have implicated the pathogenic process of COPD (14–16). Th17 cells secrete pro-inflammatory cytokines, such as interleukin (IL)-17A, IL-17F, IL-21, IL-22 and IL-23. The development of Th17 cells is dependent on retinoic acid orphan receptor (ROR)-γt expression and positively regulated by IL-6, IL-1β and IL-23 as well as transforming growth factor β (TGF-β), but negatively regulated by IL-10. Th17 cells are crucial for the pathogenesis of several inflammatory and autoimmune diseases (17, 18). Previous studies have suggested that Th17 cells may participate in the inflammatory process of COPD in animal models and humans (19–21). However, whether Th17 cells also participate in the pathogenic process of COPD in Chinese patients, who have a history of heavy smoking and exposure to dangerous air pollution, has not been clarified. CD4+CD25+Foxp3+ Tregs can inhibit inflammation (22). Treg development is dependent on the Forkhead box p3 (Foxp3) expression and is positively regulated by The Clinical Respiratory Journal (2015) • ISSN 1752-6981 2
C 2014 John Wiley & Sons Ltd V
TGF-β. Functionally, Tregs secrete inhibitory IL-10 and TGF-β. The deficiency in the number and function of Tregs is associated with the development of autoimmune diseases. However, how Tregs regulate the development of COPD remains controversial. While a higher frequency of circulating Tregs was detected in patients with COPD, along with higher percentages of Th17 cells (19), lower percentages of CD4+CD25+ T cells were detected in another population of COPD patients (23). Currently, there is no information about the frequency of circulating Tregs in Chinese patients with COPD. Notably, a previous study indicated that the imbalance of Th17 and Tregs was associated with the smokingrelated COPD in an animal model (24). Given that chronic heavy smoking causes inflammation and inhibits Treg responses, it is unclear whether the imbalance of Th17 and Tregs can exist in Chinese COPD patients, who have a history of heavy smoking and commonly expose to air pollution. Furthermore, how the degrees of Th17 and Treg imbalance are associated with the pulmonary function in those patients has not been clarified. In this study, we characterized the frequency of peripheral blood Th17 and Tregs, the relative levels of ROR-γt and Foxp3 mRNA transcripts in peripheral blood samples, and measured the levels of serum IL-17A, IL-1β, IL-6, IL-10, IL-21, IL-22, IL-23 and TGF-β in 32 Chinese patients with moderate COPD, 33 with severe COPD, 31 heavy smokers and 35 healthy controls (HC). Furthermore, we analyzed the potential association of the values of Th17, Tregs, cytokines and pulmonary function in those patients.
Materials and methods Subjects A total of 65 patients with COPD diagnosed more than 1 year, 35 smokers and 31 HC with normal pulmonary function were recruited sequentially at the inpatient and outpatient services of the Affiliated Yinzhou Hospital, College of Medicine, Ningbo University from 2010 to 2012. Individual patients with COPD were diagnosed, according to the criteria, including the 331 The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
Th17 Wangand et al.Tregs in COPD
Wang et al. Th17 and Tregs in COPD
Table 1. The demographic and clinical characteristics of participants Measure
HC (n = 31)
Smokers (n = 35)
Moderate COPD (n = 32)
Severe COPD (n = 33)
Age (year) Gender (M/F) Body mass index (kg/m2) Tobacco (pack-year) Number of admissions (time/year) LOHS (day) Use of LAMA Use of LABA Inhaled corticosteroids FEV1 (L) FEV1 % predicted FVC (L) FVC % predicted FEV1/FVC (%) WBC (×109/L) Lymphocytes (%) Neutrophils (%) CD4+ T cells (%) CD8+ T cells (%)
64 ± 8 18/15 23 ± 5.1 – – – – – – 3.3 ± 0.3 103.6 ± 7.3 4.0 ± 0.2 102.7 ± 6.7 82.3 ± 6.1 6.6 ± 1.2 30.3 ± 4.5 64.5 ± 7.2 37.0 ± 5.1 28.8 ± 4.8
67 ± 10 32/3 24.3 ± 4.9 37 (10,70) – – – – – 2.7 ± 0.2* 98.3 ± 7.7* 3.4 ± 0.15* 96.7 ± 7.8* 81.6 ± 5.8 5.9 ± 1.7 33.2 ± 3.8 63.4 ± 6.9 40.2 ± 6.8 26.4 ± 4.1
66 ± 7 26/6 24.0 ± 5.5 44 (10,82) 1.7 ± 0.7 10.4 ± 3.5 3 29 29 1.3 ± 0.2**## 53.4 ± 8.8**## 2.9 ± 0.2**## 81.4 ± 9.3**## 43.8 ± 4.4**## 6.1 ± 0.9 29.8 ± 5.1 67.8 ± 7.3 46.1 ± 6.2**## 20.6 ± 4.7**##
68 ± 7 28/5 23.6 ± 3.8 41 (18,90) 1.8 ± 0.8 13.6 ± 4.8▲▲ 2 26 26 0.7 ± 0.1**##▲▲ 36.4 ± 7.2**##▲▲ 2.7 ± 0.2**## 79.9 ± 9.6**## 26.7 ± 3.2**##▲▲ 6.5 ± 1.3 34.1 ± 3.8 60.4 ± 6.8 50.3 ± 7.5**##▲▲ 16.4 ± 3.9**##▲▲
The therapy of ICS + LABA used by COPD patients in this study is salmeterol xinafoate and fluticasone propionetae powder for inhalation or budesonide and formoterol fumarate powder for inhalation. The LAMA used by COPD patients in this study is tiotropium bromide powder for inhalation. All data expressed as mean ± standard error of the mean or median and range. *P < 0.05 and **P < 0.01 vs the HC group. # P < 0.05 and ##P < 0.01 vs the smokers. ▲ P < 0.05 and ▲▲P < 0.01 vs the moderate COPD group. CD, cluster of differentiation; COPD, chronic obstructive pulmonary disease; F, female; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; HC, healthy controls; ICS, inhaled corticosteroid; LABA, long-acting beta agonist; LAMA, long-acting muscarinic antagonist; LOHS, length of hospital stay; M, male; WBC, white blood cell.
history of tobacco smoking, clinical symptoms and impaired pulmonary function of forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) postbronchodilator
